Scientists in general chemists, physicists, biologists, biotechnologists, pathologists and clinicians in particular, need a microscope to qualitatively observe and quantitatively evaluate natural and material processes at the micro and nanometric scale.
In the area of biosensors for example, particularly in early diagnosis, it is necessary to observe and distinguish the interactions occurring, at nanometric scale, between some molecules of interest and nanoparticles. This brings to relevant results and information gathering according to the particular research in place, therefore allowing for the preventive diagnosis of specific parameters in the field of genomics, proteomics and, in general, of clinical medicine.
A nanoparticle is defined as a small object of spherical form that behaves like a single unit with regard to its transport and properties over time. Particles are generally classified according to their size, in terms of diameter, as “lame particles” from 10.000 to 2.500 nm, “fine particles” from 2.500 to 200 nm and “nanoparticles” from 200 to 1 nm.
Several highly specialized systems have been developed for nanoparticles detection and visualization. Particularly, the electron and the optical microscopy have been proven to be the most reliable techniques, leading to the development of smaller and better instrumentations over time.
Generally, optical microscopy is used for the characterization of organic samples such as cells, bacteria, erythrocytes, etcetera. Electron microscopy is commonly used for the characterization of samples such as inorganic nanoparticles, nanomaterials and single biomolecules, like proteins or DNA.
The boundary between the optical microscopy, limited by the capability to detect nanoparticles up to about 200 nanometers, and the electron microscopy, that can improve this threshold up to a few tenths of nanometers, is mainly represented by the diffraction limit described by Ernst Abbe in 1873.
Abbe's Law states that there is a fundamental limit to the resolution of any optical system and this is dictated by the diffraction of light in a optical medium. Usually, any microscope capable of capturing images with a resolution closer to this theoretical limit is named diffraction limited optical system.
The resolution of an optical microscope is mathematically defined by the Abbe's Law, corresponding to: d=0.61 λ/NA, where “d” is the size of the resolved, optically visible, object, “λ” is the wavelength of the incident light on the object and “NA” is the Numerical Aperture of the lens-objective. Therefore it is possible to obtain higher resolution images either by introducing a light source operating at lower wavelengths or by using a lens-objective having higher NA. Actually, the electron microscope reduces the diffraction limit value by generating beam of electrons, instead of photons, to excite the sample under observation. The wavelength of a beam of electrons is about few tenths of nanometers, so—according to Abbe's Law—it can lower the value of the diffraction limit to about 0.1 nm. Since the resolving power of a microscope is inversely proportional to the wavelength of the incident radiation, by using a beam of electrons it is possible to achieve a resolution better than the one obtained with an optical microscope. Even if the resolution capabilities of electron microscopes are higher than those of optical ones, the latter grant some relevant advantages, particularly in terms of cost reduction for large scale production and applications.
Thanks to the availability of superior lens-objectives and new CCD—Charge Coupled Device—cameras, optical microscopy sometimes can be more cost-effective and simpler than electron microscopy. In the last decade the use of monochromatic sources and the improvements in power and precision of the laser have increased the performances of optical microscopes to a level which is almost comparable with that bigger, more complex and expensive electron microscopes.
By localizing and concentrating a certain amount of energy in a small and well defined area of the sample under observation, the laser optical microscope aims at detecting very small particles, such as single molecules or single nanoparticles, rather than a single chain of DNA or a single protein labeled with nanoparticles. This is possible also thanks to consolidated chemical protocols of pretreatment for labeling, commonly used in many laboratories all around the world.
The possibility of recording the number of individual biomolecular events opens wide opportunities in the field of early diagnosis, aiming at detecting into the sample the presence of DNA or proteins responsible for any pathologies.
Currently, the detection of a certain DNA sequence is carried out by the use of RT PCR—Real Time Polymerase Chain Reaction—combined with molecular biology techniques such as gel electrophoresis. The PCR multiplies a specific sequence of DNA, particularly millions of copies of the same DNA sequence are generated in water solution. This is done in order to amplify, in a purely quantitative and massive manner, the signal of interest. PCR is commonly used because it is able to intercept the need of quantification of a DNA by collecting also an extremely small quantity of the sample. PCR was invented by Kary Mullis in 1993 and was developed to enhance the quantity of a genetic signal. At that time powerful video recording devices, like contemporary ones, weren't available, so there was no chance to look at the single molecule.
Since 1993, the optoelectronic technology sector has made great strides in building different types of laser microscopes able to look at the single molecule.
Any technique for detecting a single molecule involves the physical and dynamic properties of single biomolecules and allows for the indirect measure, through laser microscopes, of the fluorescence properties of chemical molecules used as biomarkers. In this way it is possible to observe biochemical processes which otherwise would not be visible using a common optical microscope. The measurement and the standardization of these processes occur thanks to the acquisition of series of optical and electrical signals from a large and heterogeneous population of biomolecules. A definition regarding the optical detection of a single molecule is given as a process that identifies the characteristics of the sample under observation, allowing to distinguish it by capturing a bright emission signal on a dark-field background with an adequate signal to noise ratio. This has already been well reported in the scientific literature from X. Michalet and S. Weiss in “Single molecule spectroscopy and microscopy” C. R Physique 3 in 2002 page. 619-644.
The possibility to detect the signal in fluorescence coming from individual molecules and nanoparticles, normally used in diagnostic tests as markers of biomolecules like DNA or proteins, is increased by the right combination between a high-sensitivity device, capable of detecting even single photons, and a low background noise. This statement is reported in the literature by X. Michalet and S. Weiss and A. N. Kapanidis, T. Laurence, F. Pinaud, S. Doose, M. Pflughoefft in “The power and prospects of fluorescence microscopies and spectroscopies,” Annu. Rev. Biophys. Biomol. Struct 32, 2003, p. 161-182.
Very often it is necessary to rely on laser fluorescence microscopy to discriminate the signal of each individual nanoparticle and this happens not only thanks to the laser power emitted on the sample, but also because of the presence of dichroic filters which are able to chromatically separate the wavelength coming from excited particles from the source emission wavelength. Moreover, for the detection of a single molecule, it is necessary to use a reduced volume of the sample, approximately few femtoliters, in order to limit the Raman scattering coming from the water molecules, which can create a high background noise. This is reported in W. E. Moerner and D. P. Fromm in “Methods of single molecule fluorescence spectroscopy and microscopy,” Rev. Sci Instrum. 74, 2004, p From 3597 to 3619.
The Confocal Microscopy is based on the use of the laser and it is suitable for studying the interface between a solid surface and air. For this reason, Confocal Microscopy is focused on the visualization of fluorescent molecules deposited on that surface. Confocal Microscopy presents some issues because the laser beam focused by an objective can excessively increase the temperature of the sample thus modifying the experiental conditions.
The Wide-field Microscopy is suitable for studying groups of molecules distributed in the section of a flow channel. However it can neither be used for the distinction of single molecules nor for the detection of nanoparticles with a size smaller than the resolution limit of the instrument. Nevertheless, it represents the basic method to reach at least micrometric vision and becomes an essential component for different kind of laser microscopy techniques which aims at seeing also at nanometric level.
TIRF Microscopy represents the most suitable and modern methodology for biosensing, TIRF Microscopy requires only a small volume of the sample under observation to identity the relevant parameters, since the portion of the total volume under analysis is limited by the penetration depth of the evanescent wave generated by total internal reflection of the laser beam in the slide coverslip. The evanescent wave illuminates both the glass slide and the aqueous solution in contact with it, with a depth of field of 200 nanometers from the glass slide into the aqueous solution. This fact considerably reduces the background noise, allowing the capture of signals from single nanoparticles localized on the surface or close to it. The limited capability of penetration of the evanescent wave in the solution represents an important benefit in biosensors since it drastically reduces the background noise, allowing the detection of nanoparticles on coverslip in real time during experiments and testing of new optical biosensors. In TIRF microscopy the generation of the evanescent wave allows to capture images and record videos of single fluorescent molecules or nanoparticles moving fast and in water solution, floating, and docking with bioreceptors printed on glass coverslip. This technique is used in the field of biosensors for the detection of labelled DNA in solution on the slide coverslip. For example, the availability in industrial scale of DNA microarray, consisting of different sequences of DNA printed on the slide coverslip, coupled with the TIRF microscopy, allows to sense and determine the phases of hybridization between complementary strands of DNA, so it can be discovered the presence or not of a specific sequence of DNA.
Laser Microscopy, thanks to the ability of detecting single molecules, offers many opportunities in the area of biomedical diagnostics, for example in the detection of small strands of DNA or in counting fluorescent nanoparticles used as DNA biomarkers.
At the state of present techniques, a Laser Microscope consists of several essential components such as light sources, mirrors, filters, objective, lenses, a camera and a XYZ manual or automated handling system, arranged all together in order to accurately handle the slide holding the sample.
The optical detection of biomolecules, that generally ranges in size from 5 nanometers, such as single strands of DNA, up to 20 nanometers, such as single proteins, occurs thanks to the attachment with covalent chemical bonds between fluorescent molecules or nanoparticles and the biomolecules of interest, according to predefined chemical protocols.
Commercial samples of spherical nanoparticles, 100 nanometers in diameter, represent an excellent compromise in terms of size either from the chemical standpoint, using them as markers of biomolecules or biomarkers, or from the physical one, since the scattering light allows the user to detect these otherwise invisible nanoparticles.
The labeled biological compound is then placed on the lens-objective of a laser microscope, built according to one of the optical techniques already patented and previously described, and the signal is detected in emission by an infinity optical system generally more or less quite complex, equipped with many components like filters, dichroic mirrors, optical switches and lenses with refined mechanisms made of durable materials like steel or aluminium.
The market availability of standard commercial nanoparticles, with the possibility to purchase any kind of element of the periodic table, delivered in a round spherical shape and characterized from the same diameter size in nanometers, will contribute to the standardization of many biosensing processes.
Nowadays there is a strong pressure towards the development of smaller, more powerful and easier to be used electronic devices. The possibility to optically detect nanoparticles with a simple and original optical method embedded into a small, lightweigth and portable device is a relevant and attractive challenge when compared with the existing expensive, and massive electron microscopes.
The laser optical systems alternative to TIRF microscopy vary depending on the target application. Atomic Force Microscopies are the most used for the study of nanomaterials dispersed into air. Confocal Microscopies are used for the study of fluorescent liquid solutions. TIRF Microscopies are widely used to study interactions between the target sample dispersed in the water layer and the receptor linked to the glass substrate.
The TIRF microscope uses a laser as light source in a total internal reflection configuration. This generates an evanescent wave able to selectively excite the nanoparticles or the single fluorescent molecules dissolved in water solution and located nearby the glass surface. This technology uses the illumination derived from evanescent wave combined with oil immersion objective having, high numerical aperture (NA>1.4) and high magnification (from 60× to 100×). In any TIRF microscope the laser beam is sent to the back focal plane of a lens-objective, out of the optical axis, and the light is focused on the pupil of that lens-objective, giving the instrument the capacity to excite what is located at the surface and to discriminate signal emitted by nanoparticles on a dark-field background. This ensures either a high degree of optical resolution or the ability to maintain the beam in total internal reflection while scanning the slide coverslip. In TIRF Microscopy the process of XY scanning of series of coverslip slides is quite complicated for the interposition of the oil, as optical medium, between the TIRF lens-objective and the slide coverslip. This is mandatory for the light beam in order to provide and maintain the total internal reflection. However, in order to generate an evanescent wave able to illuminate the surface and the nanoparticles, it is not strictly necessary that the beam is totally reflected.
For this reason, it is clear that the possibility of illuminating with a laser beam the slide coverslip, without recourring to the use of a liquid medium for optical coupling like in a oil lens-objective, could represent a unique advantage when providing optical performances slightly lower than those provided by TIRF laser microscopes in exchange of the availability of a much smaller and cheaper laser optical device.
The idea to develop a small efficient video camera for laser microscopy is based on a clever arrangement of the minimum number of required components with the purpose of minimizing size, weight and costs whilst providing adequate optical quality.
Definitely, by using an air-objective and a simple laser based excitation method it would be possible to develop a laser microscope of extremely small size, embedded into a case which is lower than 7 dm3 volume.
Moreover, the possibility of proceeding with the automatic scanning of the slide in the X and Y axis would make this mini laser microscope an optical device with an high potential for telecommunications, research and biomedical imaging.
It's important to notice that in conditions in which the force of gravity is different or even absent, the control of the image of a glass slide should be based on a air lens-objective, since the presence of oil as optical medium is not suggested in these conditions.
The gravity will not affect the liquid solution in which nanoparticles are dispersed, since the volume of the liquid under analysis is extremely small, few picoliters, and is confined in a so thin region of space between a standard microscope slide and the glass slide coverslip, thus minimizing the contribution deriving, from gravitational forces for such small nanometric samples. In optical systems based on air lens-objective, this sandwich of glass slides could be easily inserted in a slide mount in an automatic manner, via, a mechanical arm. This would allow to automate the replacement of the glass slide, accelerating the entire process of analysis for more samples.
In summary, all the already patented optical techniques previously described present optical couplings methods which are substantially different from the one presented in this patent request. Furthermore, these techniques present some critical aspects like complexity, costs, device size, device weight, high sensitivity to minor modifications due to physical misalignments, necessity of a “physical” presence in the laboratory for accurately managing oil between lens-objective and slide coverslip.
Instrumental solutions like TIRF Microscopy therefore require a keen eye combined with multidisciplinary skills of scientific level as they must continuously be subjected to a proper maintenance to provide the best reproducibility of an optical signal. TIRF Microscopy always requires the use of oil as optical medium, either using an immersion oil objective or a prism-objective. These mandatory requirements inherently induce some delays in passing from one sample to another, with the risk to incur into variability and difficulties in maintaining the accuracy of the instrument over time. This makes the whole process of laser scanning more difficult because of variations of the optical coupling between the laser, the oil and the slide coverslip. Therefore, the inner complexity of TIRF Microscopy increases its standard maintenance costs and time spent on instrument's daily maintenance.
In summary, a laser microscope is a complex and sophisticated instrument that requires an high degree of specialization and carefulness either in its design or in its operation. The proposed patent fits within the overall context described.